Up-regulated signaling from the epidermal growth factor receptor (EGFR) has been correlated with tumor invasion and metastasis in numerous human neoplasias. Recently, we have demonstrated that increased levels of EGFR promote the invasiveness of human prostate carcinoma DU-145 cells. However, the intracellular signaling pathway responsible for this enhanced tumor invasiveness has not been identified. We postulated that increased cell motility signaled via phospholipase Cgamma (PLCgamma) activation was critical for tumor invasiveness. Highly invasive DU-145 cells engineered to overexpress the EGFR were stably transfected with a dominant-negative fragment of PLCgamma from the Z-region (PLCz) or with irrelevant peptide minigenes. PLCz was expressed only in the appropriate transfectant lines, with a concomitant decrease in inositol phosphate generation. The transfectant cell lines all formed tumors when inoculated into the peritoneal cavity of athymic mice. Tumors from the cells expressing PLCz fragment were significantly less invasive than the transfectants containing the control minigenes, as assessed by the diaphragm invasion model and invasion into abdominal soft organs. The cells expressing PLCz grew and formed colonies in soft agar at rates comparable to the cells expressing the control minigenes. These data suggest that up-regulated signaling by EGFR promotes prostate tumor invasiveness secondary to increased cell motility. Furthermore, PLCgamma represents a potential therapeutic target to limit tumor progression promoted by up-regulated signaling from the EGFR and related receptors with intrinsic tyrosine kinase activity.